Using recombinant DNA technology, we have constructed a series of insertion-deletion mutants of Ha-MuSV by inserting a SalI linker oligonucleotide into various regions of Ha-MuSV genome with an occasional deletion at that region. By correlating the position of mutation with the transforming ability, we conclude that the sequence responsible for the expression of transforming ability lies within 2.5 kb from the 5' end and that the region between 0.4 kb and 1.5 kb from the 5' end contains essential sequence for transforming functions. The Ha-MuSV sequences can be rescued from the mouse cells transformed by these mutants. We have cloned the Mo-MuLV circular DNA intermediates at SalI site in lambda gtWES.lambda B-SalI vector molecules. The restriction enzyme cleavage maps have been determined for these recombinants. Two clones containing full-size Mo-MuLV genome can induce XC plaque forming Mo-MuLV. The Mo-MuLV inserts are infectious only after their excision from the vector DNA. The infectivity of the permuter DNA molecules follows two-hit kinetics, while infection with Mo-MuLV virus follows single-hit kinetics. The potential use of the cloned Mo-MuLV DNA as a vector to transfer genetic markers or to introduce foreign DNAs into mouse chromosomes will be studied.